Identifying novel amino acid substitutions of hemagglutinin involved in virulence enhancement in H7N9 virus strains

Abstract

Background: To identify site-specific features of amino acid substitutions that confer enhanced H7N9 virulence in humans, we independently generated mammalian-adapted variants of A/Anhui/1/2013 (AH-H7N9) and A/Shanghai/2/2013 (SH-H7N9) by serial passaging in Madin-Darby canine kidney (MDCK) cells.

Methods: Virus was respectively extracted from cell culture supernatant and cells, and was absolutely quantified by using real-time polymerase chain reaction. Viral RNAs were extracted and subjected to sequencing for identifying mutations. Then, site-specific mutations introduced by viral passaging were selected for further constructing HA7 or NA9 mutant plasmids, which were used to generate recombinant viruses. The interaction between the recombinant HA and receptors, H7N9-pseudotyped viruses and receptors were detected.

Results: Both subtypes displayed high variability in replicative capability and virulence during serial passaging. Analysis of viral genomes revealed multiple amino acid mutations in the hemagglutinin 7 (HA7) (A135T [AH-H7N9], T71I [SH-H7N9], T157I [SH-H7N9], T71I-V223I [SH-H7N9], T71I-T157I-V223I [SH-H7N9], and T71I-T157I-V223I-T40I [SH-H7N9]), and NA9 (N171S [AH-H7N9] and G335S [AH-H7N9]) proteins in various strains of the corresponding subtypes. Notably, quite a few amino acid substitutions indeed collectively strengthened the interactions between H7N9 strains and sialic acid receptors. Moreover, some of the amino acid substitutions identified were highly and specifically cytopathogenic to MDCK cells.

Conclusions: This study demonstrated that AH-H7N9 and SH-H7N9 subtypes can acquire enhanced receptor affinity for sialic receptors through novel amino acid substitutions. Such changes in affinitive interactions are conferred by site-specific mutations of HA7 proteins that affect the virulence and pathology of the virus strain, and/or limited compatibility between the host and the virus strain.

Keywords: Amino acid substitutions; H7N9; Hemagglutinin.

Fig. 1

Pathological changes in MDCK cells infected by various virus generations for 48 h

Fig. 2

Virus quantification. (a) Absolute RNA amounts in viruses from supernatants or cells, quantified by quantitative PCR; (b) The virulence of viruses (from supernatants or cells) in MDCK cells was quantified by measuring TCID50

Fig. 3

Interaction assessment between HA7 protein and its receptors. (a) Expression levels of the HA7 protein in 293 T cells, detected by Western blot; (b) Quantitative detection of HA binding to 3′SL- (b-1) or 6′SL-labeled receptors (b-2) by measuring KD values. (c) Quantification of NA activity was determined by measuring optical density generated in the chromogenic reaction

Fig. 4

Infectivity of recombinant H7N9 virus. (a) Quantification of viral infectivity by measuring luciferase activity; (b) Quantitative detection of the interaction between recombinant HA and 3′SL- or 6′SL-labeled receptors by measuring KD values

Fig. 5:

3D structures of interactions between the HA protein and receptors analyzed by Flusurver (https://flusurver.bii.a-star.edu.sg/INTERACTIONS/) (a) 3D structures of HA7 color-marked mutation sites and receptors; (b) Shanghai/2 HA mutation Thr40Ile and ligand NAG (pink atoms); (c) Anhui/1 HA mutation Ala135Thr and ligand GLC (pink atoms), host cell receptor SIA (pink atoms). (d) Shanghai/2 HA mutation A107T on viral chain A (yellow backbone), within 5 A from oligomeric subunit chain F (blue backbone); (e) Shanghai/2 HA mutation G218E on viral chain A (yellow backbone), within 5 A from oligomeric subunit chain C (blue backbone); (f) Shanghai/2 HA mutation T157I on viral chain A (yellow backbone), within 5 A from ligand EPE (pink atoms); (g) Shanghai/2 HA mutation V218I on viral chain A (yellow backbone), within 5 A from ligand EPE (pink atoms)

Fig. 6

Crystal structure(ID:5L14) of neuraminidase from A/Shanghai/2/2013 (H7N9) influenza virus (http://www.ebi.ac.uk/pdbe/entry/pdb/5l14/experiment). (a) 3D structures of NA9 color-marked mutation sites and receptors; (b) Anhui/1 NA mutation NA-N171S enlargement; (c) The NA-N171S mutation position (red atoms) on viral chain A (yellow backbone), within 5 A from oligomeric subunit chain B (blue backbone). (d) The NA- G335S mutation position (red atoms) on viral chain B (yellow backbone), within 5 A from oligomeric subunit chain E (blue backbone)